This invention relates to a series of substituted imidazoles, pharmaceutical compositions containing them and intermediates used in their manufacture. The compounds of the invention inhibit the production of a number of inflammatory cytokines, particularly, TNF-xcex1, and IL-1xcex2. Compounds of this invention are useful in the treatment of diseases associated with overproduction of inflammatory cytokines, such as rheumatoid arthritis, inflammatory bowel disease, septic shock, osteoporosis, and osteoarth ritis.
The inflammatory cytokines, IL-1xcex2 and TNF-xcex1 play an important role in a number of inflammatory diseases such as rheumatoid arthritis. C. Dinarello et al,. Inflammatory cytokines: Interleukin-1 and Tumor Necrosis Factor as Effector Molecules in Autoimmune Diseases Curr. Opin. Immunol. 1991, 3, 941-48. Arthritis is an inflammatory disease which affects millions of people and can strike at any joint of the human body. Its symptoms range from mild pain and inflammation in affected joints, to severe and debilitating pain and inflammation. Although the disease is associated mainly with aging adults, it is not restricted to adults. The most common arthritis therapy involves the use of nonsteroidal antiinflammatory drugs (NSAID) to alleviate the symptoms. However, despite their widespread use, many individuals cannot tolerate the doses necessary to treat the disease over a prolonged period of time. In addition, NSAIDs merely treat the symptoms of disease without affecting the underlying cause. Other drugs, such as methotrexate, gold salts, D-pencillamine, and prednisone are often used when patients fail to respond to NSAIDS. These drugs also have significant toxicities and their mechanism of action remain unknown.
Receptor antagonists to IL-1xcex2 and monoclonal antibodies to TNF-xcex1 have been shown to reduce symptoms of rheumatoid arthritis in small-scale human clinical trials. In addition to protein based therapies, there are small molecule agents which inhibit the production of these cytokines and have demonstrated activity in animal arthritis models. J. C. Boehm et al., 1-Substituted 4-Aryl-5-pyridinylimidazoles: A New Class of Cytokine Suppressive Drugs With Low 5-Lipoxygenase and Cyclooxygenase Inhibitory Potency, J. Med. Chem., 1996, 39, 3929-37. Of these small molecule agents, SB 203580 has proved effective in reducing the production of TNF-xcex1 and IL-1 in LPS stimulated human monocyte cell lines with IC50 values of 50 to 100 nM. J. Adams et al., Imidazole Derivatives And Their Use as Cytokine Inhibitor, International Patent application WO 93/14081, Jul. 23, 1993. In addition to this in vitro test, SB 203580 inhibits the production of the inflammatory cytokines in rats and mice at IC50 values of 15 to 25 mg/kg. A. M. Badger, et al, Pharmacological Profile of SB 203580, A Selective Inhibitor of Cytokine Suppressive Binding Protein/p38 Kinase, in Animal Models of Arthritis, Bone Resorption, Endotoxin Shock and Immune Function, The Journal of Pharmacology and Experimental Therapeutics, 1996, 279, 1453-61. Although human data is currently unavailable for SB 203580, monoclonal antibodies to TNF-xcex1 have proved efficacious in the treatment of rheumatoid arthritis. M. J. Elliot et al., Treatment of Rheumatoid Arthritis with Chimeric Monoclonal Antibodies to Tumor Necrosis Factor xcex1, Arthritis Rheum. 1993 36, 1681-90. Due to SB 203580""s oral activity and potency in animal models, researchers have suggested that a compound with this profile has potential as a viable treatment for rheumatoid arthritis. A. M. Badger, et al. Pharmacological Profile of SB 203580, A Selective Inhibitor of Cytokine Suppressive Binding Protein/p38 Kinase, in Animal Models of Arthritis, Bone Resorption, Endotoxin Shock and Immune Function, The Journal of Pharmacology and Experimental Therapeutics, 1996, 279, 1453-61.
SB 203580 and other small molecule agents reduce the production of inflammatory cytokines by inhibiting the activity of a serine/threonin kinase p38 (note other researchers refer to this enzyme as CSBP), at an IC50 of 200 nM. D. Griswold et al., Pharmacology of Cytokine Suppressive Anti-inflammatory Drug Binding Protein (CSPB), A Novel Stress-Induced Kinase, Pharmacology Communications, 1996, 7, 323-29. Although the precise mechanism of this kinase is unknown, it has been implicated in both the production of TNF-xcex1 and the signaling responses associated with the TNF-xcex1 receptor. 
The novel compounds of this invention inhibit the in vitro activity of p-38 in the nanomolar range. In addition, the compounds inhibit the in vitro secretion of TNF-xcex1 and IL-1xcex2 in the nanomolar range. Animal models demonstrate the inhibition of LPS induced TNF-xcex1, as well as the inhibition of rheumatoid arthritis. With this range of activity the compounds of the invention are useful in the treatment of a variety of cytokine related disorders including: rheumatoid arthritis, inflammatory bowel disease, septic shock osteoporosis, osteoarthritis, neuropathic pain, HIV replication, HIV dementia, viral myocarditis, insulin-dependent diabetes, non-insulin dependent diabetes, periodontal disease, restenosis, alopecia areta, T-cell depletion in HIV infection or AIDS, psoriasis, actue pancreatitis, allograft rejection, allergic inflammation in the lung, atherosclerosis, mutiple sclerosis, cachexia, alzheimer""s disease, stroke, Crohn""s disease, inflammatory bowel disease, ischemia, congestive heart failure, pulmonary fibrosis, hepatitis, glioblastoma, Guillain-Barre Syndrome, and systemic lupus erythematosus.
The invention relates to compounds of the Formula I 
wherein:
R1 is phenyl, substituted phenyl (where the substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl, and nitrile), or heteroaryl where the heteroaryl contains 5 to 6 ring atoms;
R2 is phenyl, substituted phenyl (where the substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl, and nitrile), heteroaryl where the heteroaryl contains 5 to 6 ring atoms and is optionally C1-4 alkyl substituted;
R3 is hydrogen, SEM, C1-5alkoxycarbonyl, aryloxycarbonyl, arylC1-5alkyloxycarbonyl, arylC1-5alkyl, substituted arylC1-5alkyl (where the aryl substituents are independently selected from one or more members of the group consisting of C1-5alkyl, C1-5alkoxy, halogen, amino, C1-5alkylamino, and diC1-5alkylamino), phthalimidoC1-5alkyl, aminoC1-5alkyl, diaminoC1-5alkyl, succinimidoC1-5alkyl, C1-5alkylcarbonyl, arylcarbonyl, C1-5alkylcarbonylC1-5alkyl, aryloxycarbonylC1-5alkyl, heteroarylC1-5alkyl where the heteroaryl contains 5 to 6 ring atoms;
R4 is xe2x80x94(A)xe2x80x94(CH2)qxe2x80x94X where:
A is vinylene, ethynylene or 
xe2x80x83where
R5 is selected from the group consisting of hydrogen, C1-5alkyl, phenyl and phenylC1-5alkyl;
q is 0-9;
X is selected from the group consisting of hydrogen, hydroxy, vinyl, substituted vinyl (where one or more substituents are selected from the group consisting of fluorine, bromine, chlorine and iodine), ethynyl, substituted ethynyl (where the substituents are selected from one or more of the group consisting of fluorine, bromine chlorine and iodine), C1-5alkyl, substituted C1-5alkyl (where the alkyl substituents are selected from the group consisting of one or more C1-5alkoxy trihaloalkyl, phthalimido and amino), C3-7cycloalkyl, C1-5alkoxy, substituted C1-5alkoxy (where the alkyl substituents are selected from the group consisting of phthalimido and amino), phthalimidooxy, phenoxy, substituted phenoxy (where the phenyl substituents are selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy), phenyl, substituted phenyl (where the phenyl substituents are selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy), arylC1-5alkyl, substituted arylC1-5alkyl (where the aryl substituents are selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy), aryloxyC1-5alkylamino, C1-5alkylamino, diC1-5alkylamino, nitrile, oxime, benzyloxyimino, C1-5alkyloxyimino, phthalitmido, succinimido, C1-5alkylcarbonyloxy, phenylcarbonyloxy, substituted phenylcarbonyloxy (where the phenyl substitutents are selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy), phenylC1-5alkylcarbonyloxy, (where the phenyl substitutents are selected from the group consisting of C1-5alkyl, halogen and C1-5alkoxy), aminocarbonyloxy, C1-5alkylaminocarbonyloxy, diC1-5alkylaminocarbonyloxy, C1-5alkoxycarbonyloxy, substituted C1-5alkoxycarbonyloxy (where the alkyl substituents are selected from the group consisting of methyl, ethyl, isopropyl and hexyl), phenoxycarbonyloxy, substituted phenoxycarbonyloxy (where the phenyl substituents are selected from the group consisting of C1-5alkyl, C1-5alkoxy, and halogen), C1-5alkylthio, substituted C1-5alkylthio (where the alkyl substituents are selected from the group consisting of hydroxy and phthalimido), C1-5alkylsulfonyl, phenylsulfonyl, substituted phenylsulfonyl (where the phenyl substituents are selected from the group consisting of bromine, fluorine, chloride, C1-5alkoxy and trifluoromethyl);
with the proviso:
if A is 
xe2x80x83q is 0 and X is H, R3 may not be SEM;
and pharmaceutically acceptable salts thereof.
In addition this invention contemplates pharmaceutical compositions containing compounds of Formula I, and methods of treating cytokine mediated disorders with compounds of Formula I.
Aside from compounds of Formula I, this invention contemplates intermediate compounds of the Formula II. These intermediates are useful in the preparation of compounds of Formula I and are as follows: 
wherein:
R1 is phenyl, substituted phenyl (where the substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl, and nitrile), or heteroaryl where the heteroaryl contains 5 to 6 ring atoms;
R2 is heteroaryl where the heteroaryl contains 5 to 6 ring atoms and is optionally C1-4alkyl substituted;
R3 is hydrogen, SEM, C1-5alkoxycarbonyl, aryloxycarbonyl, arylC1-5alkyloxycarbonyl, arylC1-5alkyl, substituted arylC1-5alkyl (where the aryl substituents are independently selected from one or more members of the group consisting of C1-5alkyl, C1-5alkoxy, halogen, amino, C1-5alkylamino, and diC1-5alkylamino), phthalimidoC1-5alkyl, aminoC1-5alkyl, diaminoC1-5alkyl, succinimidoC1-5alkyl, C1-5alkylcarbonyl, arylcarbonyl, C1-5alkylcarbonylC1-5alkyl, aryloxycarbonylC1-5alkyl, heteroarylC1-5alkyl where the heteroaryl contains 5 to 6 ring atoms;
R6 is iodine, chlorine, or bromine;
and pharmaceutically acceptable salts thereof.
In addition, this invention contemplates methods of preparing compounds of Formula I.
These methods comprise contacting a compound of Formula III 
wherein
R1 is phenyl, substituted phenyl (where the substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl, and nitrile), or heteroaryl where the heteroaryl contains 5 to 6 ring atoms;
R2 is phenyl, substituted phenyl (where the substituents are selected from the group consisting of C1-5alkyl, halogen, nitro, trifluoromethyl, and nitrile), heteroaryl where the heteroaryl contains 5 to 6 ring atoms and is optionally C1-4alkyl substituted;
R3 is hydrogen, SEM, C1-5alkoxycarbonyl, aryloxycarbonyl, arylC1-5alkyloxycarbonyl, arylC1-5alkyl, substituted arylC1-5alkyl (where the aryl substituents are independently selected from one or more members of the group consisting of C1-5alkyl, C1-5alkoxy, halogen, amino, C1-5alkylamino, and diC1-5alkylamino), phthalimidoC1-5alkyl, aminoC1-5alkyl, diaminoC1-5alkyl, succinimidoC1-5alkyl, C1-5alkylcarbonyl, arylcarbonyl, C1-5alkylcarbonylC1-5alkyl, aryloxycarbonylC1-5alkyl, heteroarylC1-5alkyl where the heteroaryl contains 5 to 6 ring atoms;
R6 is iodine, chlorine, or bromine;
with a compound of Formula IV 
where
q is 0-9 and
X is hydrogen, C1-5alkyl, substituted C1-5alkyl, hydroxy, phenyl, substituted phenyl, amino, C1-5alkylamino, nitrile, vinyl, ethynyl arylC1-5alkyl, succinimido, phthalimidooxy and halogen.
in the presence of a palladium coupling agent, a suitable solvent, and an organic base under reaction conditions which permit the preparation of a compound of Formula I.
The terms used in describing the invention are commonly used and known to those skilled in the art. However, the terms that could have other meanings are defined. The term xe2x80x9cFCSxe2x80x9d represents fetal calf serum, xe2x80x9cTCAxe2x80x9d represents trichloroacetic acid and the xe2x80x9cRPMIxe2x80x9d represents the medium from the Roswell Park Memoria Inst. (Sigma cat # R0833). xe2x80x9cIndependentlyxe2x80x9d means that when there are more than one substituent, the substitutents may be different. The term xe2x80x9calkylxe2x80x9d refers to straight, cyclic and branched-chain alkyl groups and xe2x80x9calkoxyxe2x80x9d refers O-alkyl where alkyl is as defined supra. The term heteroaryl refers to an aromatic ring of five or six members where at least one member is a heteroatom. Suitable heteroatoms include, nitrogen, oxygen and sulfur. In the case of five-membered rings the heteroaryl will contain one sulfur, oxygen, or nitrogen atom and, in addition, may contain up to three additional nitrogens. With six-membered rings the heteroaryl may contain up to three nitrogens. Examples of such heteroaryls include, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-3-yl, furan-2-yl, furan-3-yl, thiophen-2-yl, thiophen-3-yl, pyridazine, triazine, thiazole, oxazole, pyrazole and the like. xe2x80x9cSEMxe2x80x9d refers to 2-(trimethylsilyl)ethoxymethyl) and xe2x80x9cLDAxe2x80x9d refers to lithium diisopropylamide. The symbol xe2x80x9cPhxe2x80x9d refers to phenyl, xe2x80x9cPHTxe2x80x9d refers to phthalimido and the xe2x80x9carylxe2x80x9d includes mono and fused aromatic rings such as phenyl and naphthyl. The symbol C(C) represents an ethynylene group: 
and the symbol (CH)2 represents a vinylene group: 
The term xe2x80x9creaction conditionsxe2x80x9d includes physical parameters such as temperature.
As used in this invention the term xe2x80x9ccytokinexe2x80x9d refers to the proteins TNF-xcex1 and IL-1xcex2. Cytokine related disorders are diseases of humans and other mammals where the overproduction of cytokines causes the symptoms of the disease. The overproduction of the cytokines, TNF-xcex1 and IL-1xcex2 has been linked to a number of diseases. These cytokine related disorders include but are not limited to rheumatoid arthritis, inflammatory bowel disease, septic shock osteoporosis, osteoarthritis, neuropathic pain, HIV replication, HIV dementia, viral myocarditis, insulin-dependent diabetes, non-insulin dependent diabetes, periodontal disease, restenosis, alopecia areta, T-cell depletion in HIV infection or AIDS, psoriasis, actue pancreatitis, allograft rejection, allergic inflammation in the lung, atherosclerosis, mutiple sclerosis, cachexia, alzheimer""s disease, stroke, Crohn""s disease, inflammatory bowel disease, ischemia, congestive heart failure, pulmonary fibrosis, hepatitis, glioblastoma, Guillain-Barre Syndrome, and systemic lupus erythematosus. The term xe2x80x9ceffective dosexe2x80x9d refers to an amount of a compound of Formula I which reduces the amount of TNFxcex1 and/or IL-1xcex2 which may be detected in a mammal suffering from a cytokine mediated disorder. In addition, the term xe2x80x9ceffective dosexe2x80x9d refers to an amount of a compound of Formula I which reduces the symptoms of a cytokine related disorder.
The compounds of the invention may be prepared by the following schemes, where some schemes produce more than one embodiment of the invention. In those cases, the choice of scheme is a matter of discretion which is within the capabilities of those skilled in the art.
In order to produce the compounds of the invention where A is ethynylene, Scheme 1 may be used. The starting material for the scheme is a 4,5-disubstituted imidazole of the type 1a. Substituted imidazoles may be prepared following know procedures and the substituents R1 and R2 of the compounds of the invention are determined by the substituents of intermediate 1a. Intermediate 1a is treated with a base, such as NaH and an inert solvent such as DMF at room temperature for about 30 min to 1 h. Once anion formation is complete, an alkylating agent is added such as phenethyl chloride and the reaction mixture is stirred at about 60-100xc2x0 C. for about 2-4 h to give intermediates 1b1 and 1b2. These intermediates are separated at this stage to allow for the formation of final products with one predominate isomer. Although the final products may be separated, the separation of 1b1 and 1b2 leads to higher yields of products. Alternatively intermediates 1b1 and 1b2 can be prepared using the methods described in WO 96/21452, xe2x80x9cCertain 1,4,5-Trisubstituted Imidazole Compounds Useful as Cytokine.xe2x80x9d
Intermediate 1b2 is treated with a strong base such as LDA in an inert solvent such as THF at xe2x88x9278xc2x0 C. for about 30 min. A source of halogen atoms such as iodine or bromine is added to the formed anion and this mixture is allowed to warm to ambient temperature over 30 min to 1 h to give intermediate 1c where W is iodine. Treatment of 1c with a palladium coupling agent such as bis(acetatato)bis(triphenylphosphine)palladium II, a substituted ethynyl compound, such as 3-butyn-1-ol and an organic base such as triethylamine in an inert solvent such as methylene chloride at reflux gives compounds of the invention of type 1d. Alternatively, 1c may be treated with other palladium coupling agents. The agents must be palladium II entities and include but are not limited to bis(triphenylphosphine)palladiumdichloride, bis(acetonitrile)chloronitropalladium (II), bis(acetonitrile)-dichloronitropalladium (II), and bis(benzonitrile)dichloropalladium (II). In addition catalytic amounts of copper catalysts, such as copper iodide may be added to increase the speed of the reaction and/or reduce the reaction temperature from reflux to room temperature.
Although Scheme 1 is used to prepare a compound of the invention where A is ethynylene, n is 1, q is 2, X is hydroxy, R1 is 1,3-pyrimidin-4-yl, R2 is 4-chlorophenyl and R3 is phenethyl, the scheme may be used to prepare other products. For example, to vary R3, the alkylating agent, may be replaced by either another alkylating agent or an acylating agent. To prepare compounds where R3 is C1-5alkoxycarbonyl, aryloxycarbonyl, arylC1-5alkoxycarbonyl,C1-5alkylcarbonyl, and arylcarbonyl, an acylating agent replaces the benzyl chloride in Scheme 1. For example, to prepare compounds where R3 is benzoyl, benzoyl chloride relaces benzyl chloride. If compounds where R3 is substituted arylC1-5alkyl, aminoC1-5alkyl, substituted aminoC1-5alkyl and C1-5alkyl are desired, benzyl chloride may be replaced with any number of alkylating agents. For example, to prepare compounds where R3 is a substituted aminoC1-5alkyl, 1-bromo-3-dimethylaminopropane may be used is place of phenethyl chloride.
In order to vary X and q, a variety of known substituted ethynylene compounds may be used. For example if one replaces 3-butyn-1-ol with propargyl chloride, compounds where q is 1 and X is Cl, may be produced. Compounds where q is 0-9 and X is C1-5alkyl, substituted C1-5alkyl, phenyl, substituted phenyl, amino, C1-5alkylamino, nitrile, vinyl, ethynyl arylC1-5alkyl, succinimido, phthalimidooxy and halogen may all be prepared in this manner. 
Scheme 2 may be used to prepare compounds of the invention where A is vinylene. Intermediate 1a is the starting material for this scheme and is treated with a base such as NaH and an inert solvent such as DMF at room temperature for about 30 min to 1 h. Once anion formation is complete, 2-(trimethylsilyl)ethoxymethylchloride is added at room temperature and stirred for about 3-5 h to give intermediates 2a1 and 2a2. As in Scheme 1, the isomers are separated at this stage. Intermediate 2a2 is treated with a strong base such as n-butyllithium in an inert solvent such as THF at xe2x88x9278xc2x0 C. for about 1 h. A halogen source such as iodine is added and the mixture is stirred at ambient temperature for about 1 h to give intermediate 2b. Treatment of 2b with a palladium coupling agent such as bis(acetatato)bis(triphenylphosphine)palladium II, trimethylsilylacetylene and triethylamine at about 70xc2x0 C. for 18 to 24 h gives ethnyl intermediate 2c. This intermediate is treated with aqueous HBr in an alcoholic solvent such as EtOH at reflux for about 3-6 h to give a compound of Formula 1 where A is vinyl and X is Br. 
Another method of preparing compounds where A is vinylene, is illustrated by Scheme 3. The starting point for this scheme is the treatment of intermediate 2a2 with a base such as n-BuLi in an inert solvent such as THF at about xe2x88x9278xc2x0 C. under an inert atmosphere for about 15-30 min. DMF is added and this mixture is stirred at ambient temperature for about 1-5 h to give the aldehyde intermediate 3a. Treatment of 3a with Wittig reagent formed from triphenylphosphine and carbon tetrabromide, triethylamine and an inert solvent such as methylene chloride gives the vinyl compound 3b. This compound may be treated with an aqueous acid such as HCl at about room temperature over several hours to give the 2-substituted derivative 3c.
Due to the variety of known Wittig reagents, many of the compounds of the invention where A is vinyl may be prepared by Scheme 3. For example, to produce the compounds of the invention where A is vinylene, q is 1 and X is vinyl, the Wittig reagent prepared from triphenylphosphine and allyl bromide replaces the Wittig reagent used in Scheme III. Compounds where q is 1-9 and X is ethynyl, vinyl, substituted vinyl, C1-5alkyl, substituted C1-5alkyl, cycloalkyl, phenyl, araC1-5alkyl, C1-5alkylamino and nitrile may be prepared by this scheme.
In addition to compounds where A is vinylene, Scheme 3 may be used to produce compounds where A is ethynylene and X is hydroxy substituted arylalkyl. Treatment of 3c with a base such as n-BuLi in an inert solvent such as THF at xe2x88x9278xc2x0 C., followed by treatment with benzaldehyde gives the desired product 3d. 
To produce the compounds of the invention where A is 
when R5 is hydrogen, Scheme 4 may be used. Treatment of intermediate 3a with hydroxylamine in an inert solvent such as MeOH for about 3-6 h at room temperature gives intermediate 4a. The SEM group of 4a may be removed by treatment with an aqueous acid and an alcoholic solvent at reflux for about 4 h to give the desired product 4b. In order to produce the compounds of the invention where R5 is C1-5alkyl, phenyl, phenylC1-5alkyl, hydroxylamine may be replaced with the known corresponding O-substituted hydroxylamines such as O-benzylhydroxylamine. 
The compounds of the invention where X is C1-5alkylthio, substituted C1-5alkylthio, C1-5alkylsulfonyl, phenylsulfonyl and substituted phenylsulfonyl may be produced by Scheme 5. Treatment of 1c with 5-chloro-1-pentyne and a palladium coupling agent as previously described gives compound 5a. Displacement of the chloride with nucleophilic agents such as 2-mercaptoethanol in an inert solvent such as acetonitrile at room temperature gives the thiol 5b. Treatment of 5b with aqueous oxone and an inert solvent such as MeOH at ambient temperature over 3-6 h gives the sulfone compound 5c. 
To produce compounds of the invention where X is C1-5alkoxycarbonyloxy, compound 1d may be used as illustrated by Scheme 6. Treatment of compound 1d with an acylating agent such as methyl chloroformate at room temperature in an inert solvent and a mild base gives compound 6a. This method may be used to produce compounds of the invention where X is C1-5alkylcarbonyloxy, phenylcarbonyloxy, phenylC1-5alkylcarbonyloxy, aminocarbonyloxy, C1-5alkylaminocarbonyloxy, di C1-5alkylaminocarbonyloxy, C1-5alkoxycarbonyloxy, substituted C1-5alkoxycarbonyloxy, phenoxycarbonyloxy and substituted phenoxycarbonyloxy by replacing methyl chloroformate with known acylating agents. For example to prepare compounds where X is methylaminocarbonyloxy, replace methyl chloroformate with methyl isocyanate.
The compounds where X is halogen may be synthesized using 1d as illustrated by Scheme 6. Treatment of compound 1d with triphenylphosphine and a halogen source such as carbon tetrachloride at room temperature gives compound 6b. Treatment of 6b at room temperature with a nucleophilic agent such as diethyl amine gives compound 6c. 
Although the claimed compounds are useful as inhibitors of TNF-xcex1 and IL-1xcex2, some compounds are more active than others and are either preferred or particularly preferred.
The preferred compounds of Formula I include: 
The particularly preferred xe2x80x9cR1xe2x80x9ds are phenyl or substituted phenyl where the phenyl substituents are halogen or nitrile.
The particularly preferred xe2x80x9cR2xe2x80x9ds are pyrid-4-yl, pyrimidin-4-yl and 2-butyl-pyridin-4-yl.
The particularly preferred xe2x80x9cR3xe2x80x9ds are hydrogen, (CH2)3Ph and (CH2)3PHT.
The particularly preferred xe2x80x9cAxe2x80x9ds are vinylene and ethynylene.
The particularly preferred xe2x80x9cqxe2x80x9ds are 0-6.
The particularly preferred xe2x80x9cXxe2x80x9ds are hydrogen, hydroxyl, chlorine, nitrile, cyclopentyl, C1-5alkylcarbonyloxy, phenylcarbonyloxy, phenylC1-5alkylcarbonyloxy, aminocarbonyloxy, C1-5alkylaminocarbonyloxy and diC1-5alkylaminocarbonyloxy.
Compounds of Formula I may be used in pharmaceutical compositions to treat patients (humans and other primates) with disorders related to the overproduction of inflammatory cytokines, particularly TNF-xcex1. The preferred route is oral administration, however compounds may be administered by intravenous infusion or topical administration. Oral doses range from about 0.05 to 100 mg/kg, daily. Some compounds of the invention may be orally dosed in the range of about 0.05 to about 50 mg/kg daily, while others may be dosed at 0.05 to about 20 mg/kg daily. Infusion doses can range from about 1.0 to 1.0xc3x97104 xcexcg/kg/min of inhibitor, admixed with a pharmaceutical carrier over a period ranging from several minutes to several days. For topical administration compounds of Formula I may be mixed with a pharmaceutical carrier at a concentration of about 0.1 to about 10% of drug to vehicle.
The pharmaceutical compositions can be prepared using conventional pharmaceutical excipients and compounding techniques. Oral dosage forms may be elixers, syrups, capsules tablets and the like. Where the typical solid carrier is an inert substance such as lactose, starch, glucose, methyl cellulose, magnesium sterate, dicalcium phosphate, mannitol and the like; and typical liquid oral excipients include ethanol, glycerol, water and the like. All excipients may be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like using conventional techniques known to those skilled in the art of preparing dosage forms. Parenteral dosage forms may be prepared using water or another sterile carrier.
Typically the compounds of Formula I are isolated and used as free bases, however the compounds may be isolated and used as their pharmaceutically acceptable salts. Examples of such salts include hydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric, maleic, fumaric, malic, tartatic, citric, benzoic, mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic and saccharic.
The biological activity of the compounds of the invention was demonstrated by in vitro and in vivo assays. As discussed previously, agents which inhibit the activity of the enzyme p38, inhibit the production of the inflammatory cytokines TNF-xcex1, and IL-1xcex2. Compounds of the invention were measured for their ability to inhibit the activity of p38 by the following in vitro assay.
A solution (38 xcexcL) of purified recombinant p38 (where the amount of enzyme was determined empirically considering the linear range of the assay and the acceptable signal to noise ratio; 6xc3x97His-p38 expressed in E.coli), myelin basic protein substrate (also determined empirically), a buffer of pH 7.5 (Hepes:25 mM, MgCl2:10 mM, MnCl2:10 mM) were added to 92 wells of a 96-well round bottom polypropylene plate. The remaining wells were used for control (xe2x80x9cCTRLxe2x80x9d) and background (xe2x80x9cBKGxe2x80x9d). The CTRL was prepared with the enzyme, substrate buffer and 2% DMSO, and the BKG was prepared with substrate buffer and 2% DMSO. A solution (12 xcexcL) of the test compound in DMSO (compounds were diluted to 125 xcexcM in 10% DMSO/H2O and assayed at 25 xcexcM where the final DMSO concentration was 2%) was added to the testing wells. The ATP/33P-ATP solution (10 xcexcL: containing 50 xcexcM unlabeled ATP and 1 xcexcCi 33P-ATP) was added to all wells and the completed plates were mixed and incubated at 30xc2x0 C. for 30 min. Ice-cold 50% TCA/10 mM sodium phosphate (60 xcexcL) were added to each well and the plates were kept on ice for 15 min. The contents of each well were transferred to the wells of a 96-well filterplate (Millipore, MultiScreen-DP) and the filterplate was placed on a vacuum manifold, fitted with a waste collection tray. The wells were washed five times with 10% TCA/10 mM sodium phosphate (200 xcexcL) under vacuum. MicroScint-20 scintillant was added, the plates were sealed using Topseal-S sheets and counted in a Packard TopCount scintillation counter using a 33P liquid program with color quench correction, where the output is in color quench-corrected cpm. The % inhibition of the test compounds was calculated by the following formula: % inhibition=[1xe2x88x92(sample -BKG)/(CTRL-BKG)]xc3x97100.
Although compounds were initially tested at 20 xcexcM, if warranted the compounds were tested at 4-fold increments above and below that concentration. In addition, IC50s were calculated for some compounds using the Deltagraph 4-parameter curve fitting program.
Aside from the enzyme assay, many of the compounds of the invention were tested in an in vitro whole cell assay using peripheral blood mononuclear cells (xe2x80x9cPBMCxe2x80x9d) which were obtained from human blood as follows. Freshly obtained venous blood was anticoagulated with heparin, diluted with an equal volume of phosphate buffered saline (xe2x80x9cPBSxe2x80x9d) and placed in a sterile tube or other container. Aliquots (30 mL) of this mixture were transferred to centrifuge tubes which were underlaid with Ficoll-Hypaque (15 mL). The prepared tubes were centrifuged at 400xc3x97g without braking for 30 min at room temperature. Approximately xc2xd to ⅔ of the platelet layer above the mononuclear cell band was removed with a pipette. The majority of the mononuclear cell layer was carefully removed using a pipette and these PBMCs were diluted with PBS and spun at 600xc3x97g for 15 min. The resulting PBMCs were washed with another portion of PBS and spun at 400xc3x97g for 10 min at room temperature. The recovered pellets were diluted in low endotoxin RPMI/1% FCS culture medium and gave a cell concentration of 0.5-2.0xc3x97106 PMBC/mL. A small volume of the suspension was removed for counting on a hemocytometer and the remaining preparation was centrifuged at 200xc3x97g for 15 min at room temperature. The recovered pelleted PMBC were resuspended in RPMI/1% FCS to a concentration of 1.67xc3x97106/mL.
To run the assay, the PBMC suspension (180 xcexcL) was transferred to duplicate wells of a 96-well flat-bottom microtiter plate and incubated for 1 h at 37xc2x0 C. A solution of test compound (10 xcexcL: prepared at 20xc3x97 the desired final concentration) was added to each well and the plate was incubated for 1 h at 37xc2x0 C. A solution (10 xcexcL) of LPS in RPMI/1% FCS (200 ng/mL) was added and the wells were incubated overnight at 37xc2x0 C. The supernate (100 ∞L) was removed from each well and diluted with RPMI/1% FCS (400 xcexcL). The samples were analyzed for TNF-xcex1 using a commercial ELISA kit (Genzyme).
The IL-1xcex2 activity of select compounds of the invention was determined by the following in vitro assay. Plastic-adherent cells were prepared from PBMC. Briefly, PBMCs were added to the wells of a 96-well plate as above, incubated for 1 h at 37xc2x0 C., and the adherent cells prepared by gently resuspending the non-adherent cells with a pipettor, removing and discarding them and gently washing the wells 3 times with 200 xcexcL culture medium. Additional culture medium (180 xcexcL) was added to the wells after the final wash. Compound addition, LPS stimulation, incubation and supernate harvest were as for TNF-xcex1. Supernates were assayed for interleukin-1xcex2 using a commercial ELISA (Genzyme). Compounds 4 and 36 inhibited the production of IL-1xcex2 at IC50s of 7 and 13 nM respectively.
The ability of the compounds of Formula I to inhibit LPS induced TNF-xcex1 production was demonstrated in the following in vivo rodent assays. Mice (BALB/cJ females, Jackson Laboratories) or rats (Lewis males, Charles River) were fasted for 30 min prior to oral dosing with 5-10 mL/kg of test compound at 5-50 mg/kg. Thirty minutes after dosing, the animals were injected intraperitoneally with LPS at 1 mg/kg and returned to their cages for 1 h. Animals were anesthetized by CO2, exsanguinated by cardiac puncture and whole blood collected (0.1-0.7 mL). The blood was allowed to clot and serum was transferred to a centrifuge tube. This sample was centrifuged, serum was collected, aliquoted and frozen at xe2x88x9280xc2x0 C. Samples were tested by commercial ELISAs for TNF-xcex1 (Endogen for mouse TNF-xcex1 and Biosource for rat TNF-xcex1).
In addition to their in vivo TNF-xcex1 activity, a compound of Formula I inhibits polyarthritis in an in vivo rat model as follows. On day 0, male Lewis rats were injected subcutaneously near the base of the tail with 100 ul of a 7.5 mg/ml suspension of heat-killed Mycobacterium butyricum in mineral oil. Groups of rats were dosed orally, once per day, from day 0 through the end of the experiment with HCl as a negative control, or with 20 or 50 mg/kg of Cpd. 4. As a positive control for inhibition, one group was dosed with HCl on days 0-9, and then with 20 mg/kg (or 50 mg/kg)of cyclosporine (Cys) from day 10 through the end of the experiment. Under these conditions, the animals"" paws in the negative control group begin to swell on days 11-12. The paw volumes of both rear paws were determined on a mercury plesthysmograph on days 8-10, depending on the experiment, and again on days 14, 17, and either 19 or 21. The data were analyzed as the increase in paw volumes compared to the day 8-10 baseline measurements. The data obtained in four experiments is listed in Table A.
Select compounds of the invention are listed in Table B. Most compounds were tested for their ability to inhibit p38 and TNF-xcex1, however some compounds were screened in one assay. The IC50s are listed for the majority of compounds and if this calculation is unavailable, the % inhibition is listed for a given concentration. In addition to the biological data, the synthetic schemes used to prepare the compounds are listed. Since imidazoles which are unsubstituted at the 1-position are subject to tautomerization, the substituents listed for R1 and R2 are interchangeable when R3 is hydrogen.
The in vivo test results for select compounds of the invention are listed in Table C. The compounds were tested for their abity to inhibit TNF-xcex1 production in mice and/or rats and the data is listed as % inhibition at 25 mg/kg.
In order to illustrate the invention the following examples are included. These examples do not limit the invention. They are only meant to suggest a method of practicing the invention. Those skilled in the art may find other methods of practicing the invention, which are obvious to them. However those methods are deemed to be within the scope of this invention.